1. The 5th lecture for 2nd-year students
Institute for Microbiology, Medical Faculty of Masaryk University and St. Anna Faculty Hospital in Brno
Miroslav Votava
MICROBIAL BIOFILM – I
The 5th lecture for 2nd-year students
March 21, 2011

2. Resistance of microbes to drying up – revision
Very sensitive: agents of STD – gonococci,
treponemes
Less sensitive: all Gram-negative bacteria
A bit more resistant: skin flora – staphylococci,
corynebacteria
acidoresistant rods –
mycobacteria
Rather resistant: xerophiles – actinomycetes,
nocardiae, moulds
parasite cysts, helminth eggs
Highly resistant: bacterial spores

3. Practical application of water shortage
Lowering water activity stops action of most microbes → we use it for food preservation
Examples:
drying – meat, mushroom, fruit (prunes)
concentration – plum jam
salting – meat, fish, butter
sugaring – sirups, jams, candied fruit

4. The influence of heat – revision
The temperature higher than optimum → heat shock and gradual dying of cells
The number of killed cells depends on the duration of the exposure to higher temperature
The relation between the number of surviving cells and the duration of heating is inversely logarithmic one
The time needed for exterminating the whole microbial population depends on its size (on the initial number of microbes)

5. Decimal reduction time – revision
The relation between the duration of heating and the number of surviving microbes:
Log10 number
of survivors
D = decimal reduction time =
= the time required to reduce
the No of microbes to 1/10 =
= the time required to kill 90 % of
D microbes present (at the
specific temperature)
(min)

6. pH – revision
Alkalophiles: e.g. Vibrio cholerae (pH ) alkalotolerant: Proteus (it splits urea)
Acidophiles: yeasts, lactobacilli (>3), coxiellae
Some microbes are sensitive to extremes of pH: e.g. gonococci; others tolerate them, e.g Enterococcus (broad range of pH )
Low pH hinders most bacteria, namely streptococci, vibrios, putrefactive bacteria
Why sparkling water lasts longer? Because its pH is lower
Low pH keeps spores from germinating → botulism can be obtained from oil-preserved mushrooms or preserved strawberries, not from pickled gherkins or mixed pickles

7. Redox potential (rH) – revision
Aerobes – need high rH levels (>200 mV)
Anaerobes – need low rH levels (≤0 mV)
Anaerobes are killed by O2, aerobes without O2 will live
Even so, anaerobes can prosper both in nature and in our bodies – thanks to the cooperation with aerobes and facultative anaerobes (e.g. in biofilms)
Anaerobes in the body:
large intestine (99 % of bowel microorganisms) vagina oral cavity (sulci gingivales)

8. Radiation – revision UV radiation
Artificially: UV radiation is used for disinfection of surfaces, water, air; in PCR laboratories for destroying residues of DNA
Ionizing radiation (X and gamma radiation)
For sterilizing disposable syringes, infusion sets, materials for dressing and sewing, tissue grafts, some drugs, even waste and food (not in EU)
Bacterial spores are quite resistant to ionizing radiation

9. Toxic substances – revision
Their influence depends on the concentration and duration of exposure
Various microbes markedly differ in relative resistance to different types of toxic substances
In general (and contrary to drying): G– bacteria are more resistant to toxic substances than G+ bacteria (because of different structure of bacterial cell wall → presence of enzymes in periplasmatic space of G– bacteria)
For application it is vital to know the effects of the particular substances used for disinfection

10. Bacterial cell wall – revision
G G–
lipoteichoic acid O-antigen lipopoly-
inner polysaccharide saccharide
lipid A (endotoxin)
murein
porin
outer
membrane
lipoprotein
ENZYMES periplasmatic
space
inner membrane
(G–)
cytoplasmatic membrane (G+)

11. Sterilization versus disinfection – revision
Sterilization = removal of all microorganisms from objects or environment
Disinfection = removal of infectious agents from objects and environment or from the body surface
Disinfection aims at breaking the chain of infection transmission
Biocides = a new general term including also disinfectants

12. Types of disinfectants – revision
Oxidizing agents (peracetic acid, H2O2, O3)
Halogens (hypochlorite, sol. iodi)
Alkylating agents (aldehydes)
Cyclic compounds (cresol, chlorophenols)
Biguanides (chlorhexidine)
Strong acids and alkali (e.g. slaked lime)
Heavy metal compounds (Hg, Ag, Cu, Sn)
Alcohols (ethanol, propanols)
Surface active agents (QAS; e.g. cetrimid)
Others (e.g. crystal violet & other dyes)

13. Relative resistance of different agents to biocides – revision
Enveloped viruses herpesviruses
Some protozoa very susceptible Trichomonas
Gram-positive bacteria Streptococcus
Gram-negative bacteria Salmonella
Yeasts susceptible Candida
Moulds Trichophyton
Naked viruses enteroviruses
Protozoal cysts relatively resistant Giardia
Acidoresistant rods Mycobacterium
Helminth eggs Ascaris
Bacterial spores very resistant Clostridium
Coccidia Cryptosporidium
Prions extremely resistant agent of CJD
- - -

14. Two forms of microbial growth
Growth in planktonic form
Isolated microbial cells float freely in a fluid environment
Growth in biofilm form
Result of the natural tendency of microbial cells to stick to one another and to a solid surface and to form a community connected by an extracellular matter

15. Which form is more frequent?
Planktonic form
fairly common in the laboratory (e.g. in nutrient broth)
relatively scarce in a natural environment
Biofilm form
standard and crucial in the natural environment
more advantageous for microbes

16. Definition of biofilm
Microbial biofilm is a community of microorganisms that
forms at the boundary of phases (usually of the solid and fluid phase)
sticks to inert as well as to live surfaces
is surrounded by an extracellular matter, in which a complex system of channels may form

17. Three examples of biofilm
Have you ever slipped on a wet stone in a creek?
Certainly – and in was biofilm that you slipped on
Have you an aquarium and do you clean its walls?
If you do, what you wipe from them is the biofilm formed by algae
Do you clean your teeth regularly?
I hope so and by doing this you remove the biofilm called dental plaque

18. History of biofilm 1676 Antony van Leeuwehoek 1935 C. E. Zobel
bacteria in dental plaque
1935 C. E. Zobel
the first description of biofilm in marine bacteria
1950 – 1960
first information about problems with the biofilm
1978 J. W. Costerton
drawing attention to the ubiquity of biofilm
1999 Costerton, Stewart  Greenberg
biofilm involvement in persistent infections

19. Microbiology lead astray – 1
For 100 years since Pasteur and Koch times
it never occurred to anybody that in nature bacteria grow in other ways than as a freely floating plankton in seas or as colonies on agar
From the half of the 19th to the half of the 20th century,
throughout the whole „golden age of bacteriology“, the only subject of study were planktonic forms
If signs of the biofilm growth appeared the experiment was quickly „sewered“

20. Microbiology lead astray – 2
The whole microbiology has been mislead
by efforts to examine and investigate pure cultures of planktonically growing cells only, whereas the natural microbial growth is in the form of biofilm
The last area of microbiology
that started to be concerned with the biofilm is regrettably the medical microbiology, proud of its achievements with planktonic forms

21. How does the biofilm develop?
Development of biofilm = cyclic process
1. Attraction of planktonic cells to a surface
2. Adhesion of planktonic cells to the surface
3. Aggregation of cells and the development of colonies – quorum-sensing phenomenon
4. Accumulation of exopolysaccharide matrix (slime) – development of typical architecture
5. Dispersal of cells from the surface of biofilm

22. Development of biofilm – attraction
Attraction does not concern solid surfaces only
but in general the boundaries of phases
Prominent in mobile bacteria with flagella
How does the bacterium know the proximity of a surface?
It sends out chemical signals that diffuse more quickly into free areas while they concentrate in the vicinity of boundaries of phases

23. Development of biofilm – adhesion
Bacterial adhesins
fimbriae (pilli)
colonization factors of enteropathogenic E. coli
proteins and lipopolysaccharides of outer membrane
generally in most of Gram-negative bacteria
slime
both coagulase-negative and golden staphylococci
curli
E. coli

24. Development of biofilm – aggregation I
1. Movement
by means of flagella (E. coli, Vibrio cholerae)
by means of fimbriae (type IV pilli of Pseudomonas aeruginosa)
divergent – continuous layer of cells forms
convergent – aggregates develop, even of different
species (e.g. coaggregation of
Streptococcus gordonii + Fusobacterium
nucleatum in dental plaque)
2. Multiplication
both aggregation and cell division in aggregates lead to the development of microcolonies

25. Development of biofilm – aggregation II
3. Quorum sensing
During division individual cells emit chemical signals (homoserinlactones in P. aeruginosa)
After reaching a particular number of cells (quorum) the elevated concentration of signals causes the change of cellular properties:
- switching off some so far functioning genes (e.g.
a gene for the production of flagellin)
- expression of other genes, and from this
ensuing
- production of new molecules (in particular
exopolysaccharides)

26. Development of biofilm – accumulation
Production of exopolysaccharides
colanic acid (E. coli)
alginate (P. aeruginosa)
polysaccharide intercellular adhesin (Staph. epidermidis)
leads to the development of typical biofilm architecture
Its appearance depends mainly on the nature of the environment

27. Development of biofilm – dispersal
After reaching the critical amount of biomass
or after the reduction of the amount of nutrients in the environment the character of cells at the surface of biofilm changes
e.g. in P. aeruginosa the superficial cells
- cease producing alginate
- begin producing lyase and flagellin
superficial layer of biofilm starts to disintegrate
cells grow flagella and get loose of biofilm
The cells as a planktonic population drift away
to look for more suitable environment and to colonize new surfaces
The cycle closes…

28. Architecture of biofilm – I
Depends above all on the concentration of nutrients
<10 mg/L (mountain streams, lakes, open sea)
heterogeneous mosaic (a thin layer + columns of
microcolonies)
mg/L (majority of our rivers and ponds)
complex system with channels (created by mushroom-like, partially merging microcolonies)
1000 mg/L (in the environment of macroorganism)
compact biofilm (almost without traces of channels)

29. Architecture of biofilm – II
Low concentrations of nutrients (0.1 – 10 mg/L – mountain streams,
lakes, open sea)
Heterogeneous mosaic = thin layer of individual cells above which
columned microcolonies rise here and there

30. Architecture of biofilm – III
Medium concentration of nutrients (10 – 1000 mg/L – eutrophic water environment)
System with channels = mushroom-shaped microcolonies partially merging together, interwoven with water channels

31. Architecture of biofilm – IV

32. Architecture of biofilm – V
High concentrations of nutrients (>1000 mg/L – in the macroorganism)
compact biofilm = closely interconnected numerous microcolonies almost without traces of possible channels
polymicrobial = e.g. dental plaque, normal microflora of mucous membranes

33. Architecture of biofilm – VI
High concentrations of nutrients (>1000 mg/L – in the macroorganism)
compact biofilm = closely interconnected numerous microcolonies almost without traces of possible channels
b) monomicrobial = e.g. chronic osteomyelitis
biofilm on inert surfaces of medical devices

34. Architecture of biofilm – VII
Candida albicans biofilm. Alcian blue has coloured extracellular polysaccharides. Photo: Veronika Holá

35. Architecture of biofilm – VIII
Candida albicans biofilm. Toluidin blue. At the photo mushroom-like structure of the biofilm is obvious Photo: Veronika Holá

36. Properties of biofilm
Biofilm is a higher and more complex form of microbial growth
It utilizes the opportunity of mutual cooperation of cells
It enables the easier transfer of genes
It is characterized by an effective homeostasis
It shows features of a primitive circulation system
It provides a high protection against antimicrobial factors
It plays an important part in many significant occasions including medically important conditions

37. Properties of microbes in biofilm
Summary:
The properties of microbes growing in the biofilm form are fundamentally different from the properties of microbes growing in the planktonic form; the microbes
express different genes
produce different products
(extracellular matrix  flagella)
enjoy a higher degree of protection

38. Recommended reading material
Paul de Kruif: Microbe Hunters
Paul de Kruif: Men against Death
Axel Munthe: The Story of San Michele
Sinclair Lewis: Arrowsmith
André Maurois: La vie de Sir Alexander Fleming
Could you kindly supply me with another work in
connection with microbes or at least medicine?
Please mail me your suggestions at:
Thank you for your attention